The present invention relates to devices for the electrochemical detection of nucleotide sequences in fluids, to analysis cassettes for use therewith, and to methods of producing analysis cassettes.
A device for measuring ion concentrations is described in DE 41 39 121 C1, which has a measuring insert that can be inserted into a base device in an exchangeable manner. For this purpose, the fluid to be tested is placed into the measuring insert. A device for the interchangeable reception of measuring cartridges or measuring cells for the determination of biochemical measurement parameters is described in WO 98/05958. The device is connected with a computer-controlled analysis system.
In the field of medical technology, it is possible to detect various illnesses or faulty developments by detecting genetically significant traces caused by the illness or faulty development in a bodily fluid or tissue. For example, pathogenic bacteria or viruses leave behind broken pieces of their DNA (i.e. nucleotides or sequences thereof) in the blood or urine; these are nucleotide sequences, oligo-nucleotide sequences or poly-nucleotide sequences. With various types of cancerous diseases it is possible to detect portions of the diseased cancer cells with their genetically significant defects in bodily fluids. Similar types of problems arise in the field of food chemistry, where the aim is to identify the bacteria responsible for the spoilage of food by detecting corresponding genetically significant traces. The detecting of genetic traces also occurs in the course of testing food to identify source, in particular in order to obtain information whether components contained in the food stem from natural plants or genetically manipulated plants.
In accordance with a known process, blood is taken from a patient, and an anticoagulant as well as a substance which breaks open cell walls are added thereto. Added to this is a special enzyme, which cuts the DNA strands released after the cell walls have been broken open at exactly predetermined locations (“enzymatic scissors”).
Following this first step, a polymerase chain reaction (PCR) is generally performed in a second step in order to increase the sensitivity for the subsequent detection step. Such a method is described in DE 198 26 153 A1. A replication or multiplication of the DNA segments contained in the solution or fluid is stimulated by the polymerase chain reaction. The molecular elements required for this, which are the four amino acids contributing to the composition of the DNA, are added to the specimen. Then the specimen is heated in a controlled manner and cooled again thereafter. The detection process, i.e. the third step, is performed by a fluorescence-based method.
Electrochemical methods for detecting DNA segments are also known (e.g., German laid-open, non-examined Patent Application no. 199 40 647 A1, U.S. Pat. Nos. 5,312,527 and 5,871,918). Moreover, electrochemical detection methods are described in WO 01/42508 A2 and WO 95/12808. The system disclosed in the last mentioned document is very elaborate and can only be used efficiently in a large laboratory.
In accordance with electrochemical detection systems, the DNA segments or nucleotide sequences in question are detected in the solution by special working electrodes, which have been coated with special receptor molecules or biopolymers (i.e., with matching “counter pieces” to the DNA segments or nucleotide sequences in question). Only the exactly matching nucleotide sequences settle on the working electrode inserted into the fluid, so that this settling can subsequently be detected by an electrical evaluation.
A method used in this regard is PSA (potentiometric stripping analysis). In this method, a current is applied between the working electrode and a counter electrode. A slowly rising voltage can then be measured between the working electrode and a reference electrode. If nucleotide sequences of the type in question have settled on the working electrode, a stop of the voltage increase can be observed at a defined value (e.g., at 0.8 V) since at this voltage the guanine contained in the DNA segment in question, for example, is oxidized. The chronological width of this voltage plateau can be used as the measure of the amount of oxidized guanine, from which a conclusion can be drawn as to whether DNA segments of the type in question were present in a diagnostically relevant amount in the tested specimen.
The devices used with such process steps are devices used in laboratory chemistry. Because of their size, they are only suited for stationary employment. As a rule, the devices contain expensive components for transporting and metering the fluid and for the subsequent cleaning of the analysis chamber. The components include storage containers, valves, hoses, pumps, and the like. This use of such devices typically demands large space requirements. Moreover, the components require a considerable outlay for maintenance, since the demands made on cleanliness of the fluids are at a high level. Since the devices customarily used can only be used in a laboratory, respective specimens must often be transported first to a remote central laboratory. This is a great disadvantage, in particular for medical science in connection with acute illnesses.